abstract

Ciliated protozoans, such as Kyaroikeus cetarius, are a common finding in the upper respiratory tract of apparently healthy captive bottlenose dolphins, Tursiops truncatus, and the isolation of these organisms suggests they are part of the normal upper respiratory flora.^1,5 However, occasionally ciliated protozoans have been isolated from atypical locations such as the lung parenchyma, lymph nodes, and skin in animals affected by other viral, bacterial, or fungal diseases. When identified, these organisms have almost exclusively been reported as Kyaroikeus sp., although Chilodonella sp. have also been reported^1-6. In most instances, the presence of these organisms is thought to be a result of opportunistic infections or the migration of a normally commensal organism in a compromised animal. Often, the significance of the organisms relative to the overall health of the animal is unclear.^1,3-5

In July 2007, over a period of 12 days, seven out of 28 dolphins in a captive collection developed proliferative lesions in new or pre-existing superficial skin wounds. All of the affected wounds were initially unremarkable as far as size, shape, depth, color, and location. New rake or scrape wounds that became infected developed single or multiple, firm but friable, raised 3-4 mm, reddish yellow nodules within 24 hours of the appearance of the wound. Pre-existing lesions exhibited less pronounced tissue growth (<2 mm). The periphery of the lesions was raised and had a jagged appearance. Progressive erosion of this tissue resulted in an expansion of the wounds into coalescing ulcers.

The 28 animals in this collection were housed in three discrete groups with no direct physical contact among groups. Ciliates were isolated from skin lesions on dolphins in two of the three groups, including five females and two males ranging in age from 6-23 years, and 118-199 Kg. Most of the affected animals (5/7) had a history of good health with no indication of an underlying disease process that may have caused them to be immunocompromised. The sixth animal had a history of chronic recurrent pneumonia, and the seventh animal had a history of chronic lymphocytic leukemia. At the time, both of these dolphins were asymptomatic, maintaining normal appetite and behavior and otherwise appeared in good health.

Superficial skin scrapings were performed, and neutrophilic inflammation and large numbers of ciliated protozoans were observed during light microscopic examination. The organisms were oval to teardrop shaped, with elongated nuclei and the caudal two-thirds of the cell containing heavily vacuolated cytoplasm with occasional eosinophilic granules. Some six to seven days after the onset of infection, organisms undergoing binary fission were observed in the samples. These findings were consistent among all affected animals.

A skin biopsy was performed on one animal. Due to the friable nature of the tissues only epidermis and a small amount of dermal tissue were obtained. Mild edema and hypertrophy of the epidermis was noted, as well as vesicle formation with some intravesicular ciliates. Although only superficial dermal layers were present, there was extensive infiltration of these layers by neutrophils. Also, a large amount of necrotic cell debris was present, admixed with low to moderate numbers of the ciliate.

The microscopic characteristics of the ciliate were not consistent with a kyaroikeid. The features were more representative of the genus Chilodonella. Fresh samples were obtained from skin scrapings, preserved in Bouin's fixative, and submitted for further analysis using protargol (silver) impregnation staining for morphometric analysis and taxonomic classification. In addition, transmission electron microscopy was performed on fresh samples preserved in Trump's fixative. A study on the identification, development, and transmission of the ciliate is in progress.

We do not know how the organism was introduced to the dolphins, although we considered several possibilities. The enclosure is maintained as an open system, so microscopic algae, other plant matter, free swimming ciliates, and macroscopic organisms (sand fleas, shrimp, worms, and crabs) could have entered with the incoming water. Sea birds and their associated feces and scavenged food were considered as possible sources of environmental contamination. Lastly, five new animals had been added to the collection approximately six weeks prior to the outbreak, and they could have been subclinical carriers of the organism.

Because of difficulties positively identifying the organism, its life-cycle, and method of transmission, a multifaceted approach was used to attempt to eradicate the organism from the animals and the environment. Wounds were manually debrided TID and cleaned with diluted betadine, chlorhexidine or vinegar solutions. Each of these topical disinfectants proved ineffective at reducing organism numbers. Next, a topical application of zinc oxide or metronidazole gel was tried and proved unsuccessful. The two animals identified above with pre-existing conditions, and a third with rapidly expanding lesions, were treated with oral metronidazole (7 mg/kg BID) for 7-14 days to prevent the possible systemic spread of the organisms. The systemic treatment also had no apparent effect on the density of the organisms in the wounds of these animals.

Environmental control was aimed at increasing the residual levels of disinfectant in the pools to eliminate any free living stage of the organism in the environment. Incoming natural sea water for the system is passed through sand filters and disinfected with ozone prior to entering the exhibit. Due to mechanical difficulties at the time, the ozone levels could not be maintained at adequate disinfection levels. Consequently, chlorine was added to the system. However, maintaining the target concentration of 1.0-1.5 ppm in the pools proved difficult in this open system. Perhaps due to these fluctuations in concentration, the addition of chlorine also had no apparent effect on the ciliate since new cases developed during this period of increased on-contact disinfection.

Twelve days after the initial case was identified, topical cleansing TID with hydrogen peroxide (H₂O₂) was tried and an immediate reduction or elimination of the organism was observed in 6 of the seven animals. However, the ciliate was identified in a skin scrape from dolphin seven just two days prior to the onset of the H₂O₂ treatments, and large numbers of the ciliate were present for seven days after the onset of treatment. Perhaps this persistence was related to the developmental stage of the ciliate being more susceptible to the therapy. While the ciliate was present, the affected lesions failed to show any significant improvement; however, once the H₂O₂ treatments were instituted and the organisms were eliminated, the lesions in all affected animals ultimately healed unremarkably.

During the outbreak, nine skin scrapings from 6 additional dolphins and one sea lion that had open lesions (but without the characteristic changes) were examined. All samples were negative for the presence of the ciliate on light microscopic examination. At eight and 12 weeks after the initial incident, two additional cases developed in dolphins that were housed in the same pool as individuals affected during the initial outbreak. Again, lesions resolved after treatment with H₂O₂.

Future study to positively identify the organism and its life cycle is necessary to tell us more about the significance of this protozoan in T. truncatus. Full thickness skin biopsies would help establish the invasive nature of the ciliate, and may provide support for a strong causal link between the presence of the ciliate and the formation of the lesions.

References

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